Thermo Fisher Scientific Competition RED User guide

Brand: Thermo Fisher Scientific

Model: Competition RED

Type: User guide

INSTRUCTIONS

2126.0

90087 90088 90085

Competition RED Device −

Inserts and Base Plate

Number Description

90087 Competition RED Inserts, 10 pack, contains 8 dual-membrane inserts and 2 single-membrane inserts

90088 Competition RED Inserts, 50 pack, contains 40 dual-membrane inserts and 10 single-membrane inserts

90085 Competition RED Base Plate

Storage: Upon receipt store at room temperature.

Introduction

The Thermo Scientific Competition Rapid Equilibrium Dialysis (RED) Device was developed in association with

pharmaceutical laboratories to model in vivo drug interactions. The inserts used with the Competition RED Base Plate

provide an easy-to-use format for equilibrium dialysis experiments involving a small molecule and multiple binding targets.

Inserts are comprised of an O-ring-sealed vertical cylinder of dialysis membrane (MWCO ~12,000). The reusable base plate

(Product No. 90085), made of high-grade PTFE, is durable and chemically inert, eliminating nonspecific binding.

The Competition RED Device requires no extensive assembly or specialized equipment, and each chamber/well is easily

accessible from the top of the device. Additionally, the small volume and large surface area allows rapid equilibrium dialysis

within 2-4 hours with high levels of reproducibility and accuracy. The base plate enables versatile and cost-effective

experiment customization without unnecessary waste. From 4 to 16 dialysis chambers are placed into a common well, and the

multi-well format allows several experiments to be conducted simultaneously (Figure 1).

Figure 1. Format of the Thermo Scientific Competition RED Base Plate. Panel

A: The body of the Base Plate has different well sizes for holding 4, 6, 8 or 16

dialysis chambers (2, 3, 4 or 8 inserts, respectively). The different wells allow a

choice of the size best suited for the specific experimental design. Panel B: The lid

snaps onto the Base Plate and holds and suspends the dialysis chambers in the wells.

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

Important Product Information

• Each 10-pack of Competition RED Inserts contains eight dual-chamber inserts and two single-chamber inserts. The open

chamber in the single-chamber inserts enables direct access to the sample in the base plate well without disassembling

the device. An example experiment setup for monitoring drug partitioning of buffer, plasma, heart, liver, kidney and

brain tissue extracts in a single well is depicted in Figure 2. The buffer chamber is for determining the amount of

unbound drug.

Figure 2. Example experiment setup using a 6-well

compartment.

• The Competition RED Base Plate has several well sizes. Use the smallest well appropriate for the experimental design.

The volume of sample required for each well size is listed in Table 1. These volumes assume a well is completely filled

with inserts (Figure 2). Add an additional 0.5 ml to a well for each insert slot that is not filled.

Table 1. Sample volume required for each well size.

Well Total Volume*

4-compartment 1.5 ml

6-compartment 2.5 ml

8-compartment 3.0 ml

16-compartment 5.0 ml

*Add 0.5 ml for each insert slot that is not filled.

• Tissue extract preparation can influence results of equilibrium dialysis experiments. Use the best practices available and

use similar procedures when preparing samples for each experiment. General tissue-handling precautions are as follows:

do not over-homogenize; dilute extracts 10-fold with an isotonic buffer; and use wide-bore pipette tips.

Additional Materials Required

• Reusable Competition RED Base Plate (Product No. 90085)

• Isotonic dilution buffer: for example, phosphate-buffered saline (PBS) containing 100 mM sodium phosphate and

150 mM sodium chloride (Product No. 28372)

• Sealing Tape for 96-Well Plates (Product No. 15036)

• 20% ethanol

• Shaker and incubator

Procedure for Competition Equilibrium Dialysis

A. Prepare the Base Plate

1. Rinse the base plate wells and lid with 20% ethanol for 10 minutes.

2. Remove ethanol and rinse twice with ultrapure water.

3. Allow plate to dry. Use plate immediately or store covered.

Note: The inserts are supplied ready to use for dialysis. Rinsing the insert is unnecessary; however, if you want to rinse

the inserts, refer to the Appendix.

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

B. Equilibrium Dialysis

The following example protocol is for monitoring the partitioning of a drug between plasma and tissue samples. Optimize or

modify this procedure as required for specific applications and analysis methods.

1. Determine the number of binding targets (such as tissue extracts) to be examined in a competition dialysis experiment

and the corresponding number of inserts and well size required (see Figure 2 in the Important Product Information

section for an example experimental setup).

2. Prepare 10-fold (w/v) tissue homogenates diluted in isotonic dilution buffer. Each dialysis chamber requires 200 μl of

extract. Duplicates can be performed in the same well or in a duplicate experiment in another well.

Note: If performing precipitation to prepare sample for analysis (see Procedure for Sample Analysis – For Unlabeled

Compounds, Step 5) prepare an additional 100 μl of extract.

3. For each test compound, prepare the required volume and concentration by mixing the test compound with plasma

diluted 10-fold with isotonic dilution buffer. The volume required is based on the base plate well size (Table 1). For

tissue binding studies, a drug concentration of 1 μM is common.

Note: If performing precipitation to prepare sample for analysis (see Procedure for Sample Analysis – For Unlabeled

Compounds, Step 4), 50 μl of diluted plasma free of test compound is required for each tissue and buffer sample.

4. Transfer the test compound/plasma mixture prepared in Step 3 to an appropriately sized well. Place lid onto the base plate.

5. Suspend the dialysis chambers (open end up) into the well of the base plate containing the test compound by placing

inserts into the appropriate slots of the base plate lid. To avoid damage, do not touch the dialysis membrane.

6. Place 200 μl of tissue extract, buffer or alternative binding target into a dialysis chamber, which is indicated by a red

ring. Record the appropriate location of each binding target.

7. Cover the unit with sealing tape and incubate at 37°C on an orbital shaker at 100-500 rpm.

Note: Incubating for 4 hours is generally sufficient to achieve equilibrium; however, actual time required may differ

depending on the test compounds and shaker used. For best results, perform a pilot experiment to empirically determine

the time required to reach equilibrium before processing samples. Time periods greater or less than 4 hours can be used;

however, excessively long incubations (i.e., ≥ 6 hours) may promote instability of extracts or test compounds, or result in

a volume change from hydrostatic pressure.

8. Remove equal volumes from each of the dialysis chambers and from the bottom well and place them in separate

microcentrifuge tubes or into a deep-well plate for analysis. Use an insert with a open side (lacking a dialysis membrane)

to directly access the bottom well.

9. Remove and discard used inserts and wash the base plate for reuse.

Note: The inserts can be easily removed with forceps or the Thermo Scientific RED Device Insert Removal Tool

(Product No. 89812), which enables quick removal of eight inserts at once. Alternatively the base plate lid can be

removed and inserts pushed up from the bottom.

Procedure for Sample Analysis

Determine the test compound concentration in the plasma, buffer and tissue samples to determine the in vitro partitioning

ratios (Kp). Some common analysis methods include LC/MS/MS, radioactivity and UV/visible/fluorescent spectrometry.

Modify the example protocols below as required.

• Procedure for radio-labeled compounds

1. Remove 50 μl of each sample and place into a scintillation vial appropriate for available instrumentation. Follow

manufacture’s recommendations for determining total radioactivity counts per sample.

2. Use the ratio of radio counts to determine the in vitro Kp value between the tissue and the plasma. The free drug fraction

can also be obtained from a ratio of the buffer and plasma.

sample plasma of counts radio sample tissue of counts radio

Kp =

100% x

sample plasma of counts radio sample buffer of counts radio

drug Free =

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

• Procedure for unlabeled compounds

Note: For unlabeled compounds, place the samples in a common matrix and then treat with precipitation buffer (Step 1)

containing an internal standard (IS) before analysis by LC/MS/MS using the protocol below. For fast processing of many

samples, use Thermo Scientific Pierce Protein Precipitation Plates (Product No. 90036).

1. Prepare precipitation buffer (e.g., cold 90/10 acetonitrile/water with 0.1% formic acid) containing an internal standard.

Each sample requires 600 μl of precipitation buffer.

2. Pipette 50 μl each of the post-dialysis tissue and buffer samples into separate microcentrifuge tubes.

3. Transfer 50 μl each of post-dialysis plasma sample into separate microcentrifuge tubes equal to the number of tissues

and buffer samples from Step 2.

4. To each post-dialysis tissue or buffer sample from step 2 add 50 μl of plasma diluted 10-fold with isotonic buffer (i.e.,

pre-dialysis starting sample minus drug). This creates a consistent matrix between samples to correct for possible

variations during precipitation.

5. To the post-dialysis plasma samples from Step 3, add 50 μl of a corresponding tissue extract (i.e., pre-dialysis starting

sample) to create a consistent matrix between samples to correct for possible variations during precipitation.

Note: Refer to Table 2 for an example of steps 3-5 for the experiment represented in Figure 2.

Table 2. Example precipitation setup.

Tube # Sample (50 μl) Matrix (50 μl)

1 Post-dialysis liver extract 10X diluted plasma

2 Post-dialysis heart extract 10X diluted plasma

3 Post-dialysis kidney extract 10X diluted plasma

4 Post-dialysis brain extract 10X diluted plasma

5 Post-dialysis buffer 10X diluted plasma

6 Post-dialysis plasma liver extract

7 Post-dialysis plasma heart extract

8 Post-dialysis plasma kidney extract

9 Post-dialysis plasma brain extract

10 Post-dialysis plasma isotonic buffer

6. Add 300 μl of precipitation buffer to each tube. Vortex and incubate for 30 minutes on ice. Precipitation of the proteins

in the sample releases free and bound drug for analysis.

7. Centrifuge for 10 minutes at 13,000-15,000 × g.

8. Transfer supernatant to a vial or plate for quantitative measurements by LC/MS/MS.

Note: If final sample is too dilute, dry and reconstitute it in a smaller volume before analysis.

9. Determine the concentration of test compound in the buffer and plasma chambers from peak areas relative to the internal

standard.

10. After LC/MS/MS analysis, calculate the in vitro Kp value as follows:

IS against ratio count plasma IS against ratio count sample

Kp =

100% x

IS against ratio count plasma IS against ratio count buffer

drug Free =

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

Appendix

A. Rinsing the RED Device Inserts (optional)

The RED Device Inserts are supplied ready to use for dialysis with plasma and buffer. Rinsing the insert is unnecessary;

however, if you want to rinse the inserts, use the following protocol.

1. Soak the number of required Competition RED Device Inserts in ultrapure water for 10 minutes.

2. Discard water and soak for 10 minutes. There is no need to remove water from individual inserts between soaking steps.

3. Store inserts in ultrapure water before use. Do not allow the membranes to dry after rinsing. If required, store inserts in

water at 4-8°C for up to 1 week.

4. Before use remove water by inverting and shaking gently.

Related Thermo Scientific Products

15036 Sealing Tape for 96-Well Plates, 100/pkg

28372 BupH™ Phosphate Buffered Saline Packs, 40 packs

51101 Acetonitrile, 1 L

28904 Trifluoroacetic Acid, Sequanal grade, 10 × 1 ml

90036 Pierce Protein Precipitation Plates

89809 RED Device Inserts, 50/pk

90004 Single-Use RED Base Plate (empty), 2 plates

RED Device Products are manufactured exclusively for Thermo Fisher Scientific by Linden Bioscience.

U.S. and international patent pending on RED Device by Linden Bioscience.

This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale,

as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and

workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications

regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the

Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This

warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).

No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular

purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or

refund for the non-conforming Product(s).

There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii)

use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products.

Current versions of product instructions are available at www.thermo.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.

subsidiaries. Printed in the USA.